Cyanidin and cyanidin 3-O-β-D-glucoside as DNA cleavage protectors and antioxidants

Anthocyanins, colored flavonoids, are water-soluble pigments present in the plant kingdom; in fact they are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. Present in beans, fruits, vegetables and red wines, considerable amounts of anthocyanins are ingested as constituents of the human diet (180-215 mg daily). There is now increasing interest in the in vivo protective function of natural antioxidants contained in dietary plants against oxidative damage caused by free radical species. Recently, the antioxidant activity of phenolic phytochemicals, has been investigated. Since the antioxidant mechanism of anthocyanin pigments is still controversial, in the present study we evaluated the effects of cyanidin and cyanidin 3-O-beta-D-glucoside on DNA cleavage, on their free radical scavenging capacity and on xanthine oxidase activity. Cyanidin and cyanidin 3-O-beta-D-glucoside showed a protective effect on DNA cleavage, a dose-dependent free radical scavenging activity and significant inhibition of XO activity. These effects suggest that anthocyanins exhibit interesting antioxidant properties, and could therefore represent a promising class of compounds useful in the treatment of pathologies where free radical production plays a key role.     

Effects of dietary antioxidants on human DNA ex vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and alpha-tocopherol, were investigated, at concentrations up to 200 microM, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 min exposure to hydrogen peroxide (final concentrations H2O2: 30, 45, or 60 microM). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H2O2 from these polyphenolics. Neither ascorbic acid nor alpha-tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.     

Separation procedures for naturally occurring antioxidant phytochemicals

Phytochemicals in fruits, vegetables, spices and traditional herbal medicinal plants have been found to play protective roles against many human chronic diseases including cancer and cardiovascular diseases (CVD). These diseases are associated with oxidative stresses caused by excess free radicals and other reactive oxygen species. Antioxidant phytochemicals exert their effect by neutralizing these highly reactive radicals. Among the tens of thousands of phytochemicals found in our diets or traditional medicines, polyphenols and carotenoids stand out as the two most important groups of natural antioxidants. However, although collectively these phytochemicals are good antioxidants, the roles and effect of individual compounds are often not well known. Hundreds of carotenoids and thousands of polyphenols have been identified so far from various plants. A single plant could contain highly complex profiles of these compounds, which sometimes are labile to heat, air and light, and they may exist at very low concentrations in the plants. This makes the separation and detection of these antioxidant phytochemicals a challenging task. The present review focuses on the antioxidant activity, chemical types, sampling and sample processing procedures, and separation using various chromatographic and electrophoretic techniques. Detection and quantification using ultraviolet-visible-diode array and mass spectrometry will be discussed.     

Effects of Dietary Antioxidants on Human DNA Ex Vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and &#102 -tocopherol, were investigated, at concentrations up to 200 &#117 &#119 M, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 &#117 min exposure to hydrogen peroxide (final concentrations H 2 O 2 : 30, 45, or 60 &#117 &#119 M). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H 2 O 2 from these polyphenolics. Neither ascorbic acid nor &#102 -tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.     

The protective effect and mechanism of soybean oil and its extracts on DNA damage in human ECV304 cells exposed to UV-C

The degree of DNA damage in the human endothelial cell line ECV304 exposed to UV-C, with or without the presence of soybean oil (SBO), was assessed by the Comet assay. After 5-min exposure to UV-C, the %Tail DNA in the ECV304 cells ranged from 0% to 20% for SBO treatment groups and from 50% to 70% for the control group. The result indicated a strong protective effect of SBO against UV-C-induced DNA damage. To clarity the mechanism of this protective effect of SBO, the methanol extract of SBO (MESO) was analyzed and its capacity against UV-C-induced DNA damage was evaluated. Gas chromatography mass spectrometry (GC-MS) analysis confirmed that MESO contained many antioxidants including n-3-polyunsaturated fatty acid (n-3-PUFA), tocopherols and phytosterols. Comet assay revealed that the MESO was also active in reducing the DNA damage dose-dependently (P<0.0001) vs. control in the ECV304 cells. Therefore, we concluded that these potential antioxidants may be responsible for the scavenge of oxidative radicals induced by UV-C irradiation. This study suggested that dietary SBO, which is abundant of antioxidants, may reduce the content or impact of reactive oxygen species (ROS) and lower the risk of diseases caused by ROS.     

An Investigation of the Antioxidant Activity of Black Tea Using Enhanced Chemiluminescence

Antioxidants are important species which possess the ability to protect the body from damage caused by free radical-induced oxidative stress. A variety of free radical-scavenging antioxidants exist within the body many of which are derived from dietary sources. There is currently much interest in the antioxidant role of flavonoids and other polyphenols found in tea, wine, fruit and vegetables. Enhanced chemiluminescence is a simple technique which can be used as a rapid and sensitive assay for measuring the antioxidant activity of beverages such as green and black tea. This article examines the impact of water temperature, stewing time, leaf concentration and the addition of milk upon the antioxidant activity of black tea solutions. The antioxidant activity of a range of commercially available black and green teas has also been measured.     

Potent antioxidant dendrimers lacking pro-oxidant activity

It is well known that antioxidants have protective effects against oxidative stress. Unfortunately, in the presence of transition metals, antioxidants, including polyphenols with potent antioxidant activities, may also exhibit pro-oxidant effects, which may irreversibly damage DNA. Therefore, antioxidants with strong free radical-scavenging abilities and devoid of pro-oxidant effects would be of immense biological importance. We report two antioxidant dendrimers with a surface rich in multiple phenolic hydroxyl groups, benzylic hydrogens, and electron-donating ring substituents that contribute to their potent free radical-quenching properties. To minimize their pro-oxidant effects, the dendrimers were designed with a metal-chelating tris(2-aminoethyl)amine (TREN) core. The dendritic antioxidants were prepared by attachment of six syringaldehyde or vanillin molecules to TREN by reductive amination. They exhibited potent radical-scavenging properties: 5 times stronger than quercetin and 15 times more potent than Trolox according to the 1,1-diphenyl-2-picrylhydrazyl assay. The antioxidant dendrimers also protected low-density lipoprotein, lysozyme, and DNA against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced free radical damage. More importantly, unlike quercetin and Trolox, the two TREN antioxidant dendrimers did not damage DNA via their pro-oxidant effects when incubated with physiological amounts of copper ions. The dendrimers also showed no cytotoxicity toward Chinese hamster ovary cells.     

Protective effects of green tea polyphenols and their major component, (-)-epigallocatechin-3-gallate (EGCG), on 6-hydroxydopamine-induced apoptosis in PC12 cells

Green tea polyphenols exert a wide range of biochemical and pharmacological effects, and have been shown to possess antimutagenic and anticarcinogenic properties. Oxidative stress is involved in the pathogenesis of Parkinson's disease. However, although green tea polyphenols may be expected to inhibit the progression of Parkinson's disease on the basis of their known antioxidant activity, this has not previously been established. In the present study, we evaluated the neuroprotective effects of green tea polyphenols in the Parkinson's disease pathological cell model. The results show that the natural antioxidants have significant inhibitory effects against apoptosis induced by oxidative stress. 6-Hydroxydopamine (6-OHDA)-induced apoptosis in catecholaminergic PC12 cells was chosen as the in vitro model of Parkinson's disease in our study. Apoptotic characteristics of PC12 cells were assessed by MTT assay, flow cytometry, fluorescence microscopy and DNA fragmentation. Green tea polyphenols and their major component, EGCG at a concentration of 200 microM, exert significant protective effects against 6-OHDA-induced PC12 cell apoptosis. EGCG is more effective than the mixture of green tea polyphenols. The antioxidant function of green tea polyphenols may account for this neuroprotective effect. The present study supports the notion that green tea polyphenols have the potential to be effective as neuropreventive agents for the treatment of neurodegenerative diseases.     

Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans

The beneficial effects of polyphenol compounds in fruits and vegetables are mainly extrapolated from in vitro studies or short-term dietary supplementation studies. Due to cost and duration, relatively little is known about whether dietary polyphenols are beneficial in whole animals, particularly with respect to aging. To address this question, we examined the effects of blueberry polyphenols on lifespan and aging of the nematode, Caenorhabditis elegans, a useful organism for such a study. We report that a complex mixture of blueberry polyphenols increased lifespan and slowed aging-related declines in C. elegans. We also found that these benefits did not just reflect antioxidant activity in these compounds. For instance, blueberry treatment increased survival during acute heat stress, but was not protective against acute oxidative stress. The blueberry extract consists of three major fractions that all contain antioxidant activity. However, only one fraction, enriched in proanthocyanidin compounds, increased C. elegans lifespan and thermotolerance. To further determine how polyphenols prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. Prolonged lifespan from this treatment required the presence of a CaMKII pathway that mediates osmotic stress resistance, though not other pathways that affect stress resistance and longevity. In conclusion, polyphenolic compounds in blueberries had robust and reproducible benefits during aging that were separable from antioxidant effects.     

Protective effects of acerola juice on genotoxicity induced by iron in vivo

Metal ions such as iron can induce DNA damage by inducing reactive oxygen species (ROS) and oxidative stress. Vitamin C is one of the most widely consumed antioxidants worldwide, present in many fruits and vegetables, especially inMalpighia glabra L., popularly known as acerola, native to Brazil. Acerola is considered a functional fruit due to its high antioxidant properties and phenolic contents, and therefore is consumed to prevent diseases or as adjuvant in treatment strategies. Here, the influence of ripe and unripe acerola juices on iron genotoxicity was analyzed in vivo using the comet assay and micronucleus test. The comet assay results showed that acerola juice exerted no genotoxic or antigenotoxic activity. Neither ripe nor unripe acerola juices were mutagenic to animals treated with juices, in micronucleus test. However, when compared to iron group, the pre-treatment with acerola juices exerted antimutagenic activity, decreasing significantly micronucleus mean values in bone marrow. Stage of ripeness did not influence the interaction of acerola compounds with DNA, and both ripe and unripe acerola juices exerted protective effect over DNA damage generated by iron.     

Evaluation of DNA damage in flight personnel by Comet assay

There have been some suggestions that air-crew are at a higher-than-normal risk of developing cancer, since they are exposed to potential genotoxic factors. These include cosmic radiations, airborne pollutants such as the combustion products of jet propulsion, ozone, and electromagnetic fields. We used the Comet assay to investigate DNA damage in flight personnel with the aim of assessing potential health hazards in this occupational category. We studied 40 civil air-crew members who had been flying long-haul routes for at least 5 years, and compared them with a homogeneous control group of 40 healthy male ground staff. The Comet assay, or single-cell gel electrophoresis (SCGE), detects DNA single- and double-strand breaks (DSBs) and alkali-labile lesions in individual cells, and is a powerful and sensitive technique for detecting genetic damage induced by different genotoxic agents. Taking into consideration occupational risk and possible confounding factors, this assay showed a small increase, that did not reach statistical significance, of DNA damage in long-haul crew members compared to controls, indicating a lack of evident genotoxic effects. An association, although again not statistically significant, was found between reduced DNA damage and use of protective drugs (antioxidants).     

In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L

Recent evidence shows that plant polyphenols exhibit antioxidant and radical scavenging properties. By three separate and complementary methods--DPPH assay, beta-carotene-linoleic acid assay and NBT-reduction assay it was established that a polyphenol-rich extract from the medicinal plant Geranium sanguineum L. with strong anti-influenza virus activity, possessed antioxidant and radical scavenging capacities. For comparative reasons caffeic acid and the synthetic antioxidant BHT were used. Total soluble phenolic constituents of the MeOH extract measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). Further it was demonstrated that the EtOAc fraction, retaining the majority of the in vivo protective effect exhibited a strong O2-scavenging activity while the n-BuOH fraction, containing the majority of the in vitro antiviral activity provoked generation of O2-. The O2- scavenging activity of all three preparations correlated with the rate of the protective effect shown in the murine model of experimental influenza virus infection. The present results are in accordance with our intensive studies on the mode of the protective effect of the plant extract which showed positively that the protection may possibly be attributed to the combination of more than one biological activities and that the use of antioxidants might be an useful approach in the treatment of influenza infection.     

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.     

Role of Nitric Oxide Synthases in Parkinson’s Disease: A Review on the Antioxidant and Anti-inflammatory Activity of Polyphenols

Natural polyphenols can exert protective action on a number of pathological conditions including neurodegenerative disorders. The neuroprotective effects of many polyphenols rely on their ability to permeate brain barrier and here directly scavenge pathological concentration of reactive oxygen and nitrogen species and chelate transition metal ions. Importantly, polyphenols modulate neuroinflammation by inhibiting the expression of inflammatory genes and the level of intracellular antioxidants. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by several abnormalities including inflammation, mitochondrial dysfunction, iron accumulation and oxidative stress. There is considerable evidence showing that cellular oxidative damage occurring in PD might result also from the actions of altered production of nitric oxide (NO). Indeed, high levels of neuronal and inducible NO synthase (NOS) were found in substantia nigra of patients and animal models of PD. Here, we evaluate the involvement of NOS/NO in PD and explore the neuroprotective activity of natural polyphenol compounds in terms of anti-inflammatory and antioxidant action. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Effect of the phytoestrogen, genistein-8-C-glucoside, on Chinese hamster ovary cells in vitro

Genistein-8-C-glucoside (G8CG) belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. A number of studies on flavonoids show their cardioprotective and antiosteoporosis properties in in vitro and in vivo models. As a phytoestrogen, genistein has recently generated interest as a potential anticancer and antiatherogenic agent. Several flavonoids are known as antioxidants and scavengers of free oxygen radicals. In the current investigation we used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.). Many authors have found that the action of genistein is not so simple, although many reports conducted in vitro have demonstrated that it is cytotoxic and genotoxic. Therefore, the cytotoxic and genotoxic effects of this compound in Chinese hamster ovary cells (line CHO) were studied. A colorimetric MTT assay to assess cytotoxicity and a Comet assay for the detection of DNA damage were used. Apoptosis was determined by the Hoechst 33258/propidium iodide staining technique. We have also demonstrated antioxidant properties of G8CG. The level of reactive oxygen species generated by G8CG alone and/or H2O2 was evaluated with fluorescence probes: dichlorofluorescein-diacetate (DCFDA) by flow cytometry. The cells were exposed to various concentrations of genistein-8-C-glucoside (1-290 microM) and hydrogen peroxide (10-130 microM) and the effect of G8CG alone or in combination with H2O2 was determined. The results reveal that G8CG at concentrations higher than 10 microM significantly reduced cell viability, induced apoptosis and DNA damage. However at lower concentrations (5 and 7.5 microM), G8CG showed antioxidant properties, but had no cytotoxic or genotoxic activity.     

Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay

Antioxidant species may act in vivo to decrease oxidative damage to DNA, protein and lipids thus reducing the risk of coronary heart disease and cancer. Phytoestrogens are plant compounds which are a major component of traditional Asian diets and which may be protective against certain hormone-dependent cancers (breast and prostate) and against coronary heart disease. They may also be able to function as antioxidants, scavenging potentially harmful free radicals. In this study, the effects of the isoflavonoids (a class of phytoestrogen) genistein and equol on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide significantly increased the levels of DNA strand breaks. Pre-treatment of the cells with both genistein and equol offered protection against this damage at concentrations within the physiological range. This protection was greater than that offered by addition of the known antioxidant vitamins ascorbic acid and alpha-tocopherol, or the compounds 17beta-oestradiol and Tamoxifen which have similar structures to isoflavonoids and are known to have weak antioxidant properties. These findings are consistent with the hypothesis that phytoestrogens can, under certain conditions, function as antioxidants and protect against oxidatively-induced DNA damage.     

Role of plant polyphenols in genomic stability

Polyphenols are a large and diverse class of compounds, many of which occur naturally in a range of food plants. The flavonoids are the largest and best-studied group of these. A range of plant polyphenols are either being actively developed or currently sold as dietary supplements and/or herbal remedies. Although, these compounds play no known role in nutrition (non-nutrients), many of them have properties including antioxidant, anti-mutagenic, anti-oestrogenic, anti-carcinogenic and anti-inflammatory effects that might potentially be beneficial in preventing disease and protecting the stability of the genome. However not all polyphenols and not all actions of individual polyphenols are necessarily beneficial. Some have mutagenic and/or pro-oxidant effects, as well as interfering with essential biochemical pathways including topoisomerase enzyme activities, prostanoid biosynthesis and signal transduction. There is a very large amount of in vitro data available, but far fewer animal studies, and these are not necessarily predictive of human effects because of differences in bacterial and hepatic metabolism of polyphenols between species. Epidemiological studies suggest that high green tea consumption in the Japanese population and moderate red wine consumption in the French population may be beneficial for heart disease and cancer, and these effects may relate to specific polyphenols. A small number of adequately controlled human intervention studies suggest that some, but not all polyphenol extracts or high polyphenol diets may lead to transitory changes in the antioxidative capacity of plasma in humans. However, none of these studies have adequately considered long-term effects on DNA or the chromosome and unequivocally associated these with polyphenol uptake. Furthermore, clinical trials have required intravenously administered polyphenols at concentrations around 1400mg/m(2) before effects are seen. These plasma concentrations are unlikely to be achieved using the dietary supplements currently available. More focused human studies are necessary before recommending specific polyphenolic supplements at specific doses in the human population.     

How do nutritional antioxidants really work: Nucleophilic tone and para-hormesis versus free radical scavenging in vivo

We present arguments for an evolution in our understanding of how antioxidants in fruits and vegetables exert their health-protective effects. There is much epidemiological evidence for disease prevention by dietary antioxidants and chemical evidence that such compounds react in one-electron reactions with free radicals in vitro. Nonetheless, kinetic constraints indicate that in vivo scavenging of radicals is ineffective in antioxidant defense. Instead, enzymatic removal of nonradical electrophiles, such as hydroperoxides, in two-electron redox reactions is the major antioxidant mechanism. Furthermore, we propose that a major mechanism of action for nutritional antioxidants is the paradoxical oxidative activation of the Nrf2 (NF-E2-related factor 2) signaling pathway, which maintains protective oxidoreductases and their nucleophilic substrates. This maintenance of “nucleophilic tone,” by a mechanism that can be called “para-hormesis,” provides a means for regulating physiological nontoxic concentrations of the nonradical oxidant electrophiles that boost antioxidant enzymes, and damage removal and repair systems (for proteins, lipids, and DNA), at the optimal levels consistent with good health.